CDK4/6 inhibition enhances T-cell immunotherapy on hepatocellular carcinoma cells by rejuvenating immunogenicity

Hepatocellular carcinoma (HCC) poses a significant clinical challenge, necessitating the integration of immunotherapeutic approaches. Palbociclib, a selective CDK4/6 inhibitor, has demonstrated promising efficacy in preclinical HCC models and is being evaluated as a novel therapeutic option in clinical trials. Additionally, CDK4/6 inhibition induces cellular senescence, potentially influencing the tumor microenvironment and immunogenicity of cancer cells. In this study, we conducted comprehensive bioinformatic analyses using diverse HCC transcriptome datasets, including bulk and single-cell RNA-sequencing data from public databases. We also utilized human and mouse HCC cells to investigate functional aspects. Primary T cells isolated from mouse blood were employed to assess T cell immunity against HCC cells. Results revealed that CD8+ T-cell infiltration correlates with improved outcomes in HCC patients with suppressed CDK4/6 expression. Moreover, CDK4/6 expression was associated with alterations in the immune landscape and immune checkpoint expression within the liver tumor microenvironment. Furthermore, we found that treatment with Palbociclib and Doxorubicin induces cellular senescence and a senescence-associated secretory phenotype in HCC cells. Notably, pretreatment with Palbociclib augmented T cell-mediated cytotoxicity against HCC cells, despite upregulation of PD-L1, surpassing the effects of Doxorubicin pretreatment. In conclusion, our study elucidates a novel mechanism by which CDK4/6 inhibition enhances T-cell-associated cancer elimination and proposes a potential therapeutic strategy to enhance T-cell immunotherapy on HCC. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12935-024-03351-z.


Introduction
Hepatocellular carcinoma (HCC) stands as a leading cause of cancer-related mortality on a global scale [1].The HCC incidence is attributed to diverse risk factors, including chronic viral hepatitis, fatty liver disease, alcohol-related liver cirrhosis, smoking, obesity, diabetes, iron overload, and various dietary exposures, all of which lead to hepatic injury, cirrhotic progression, and inflammation, causing hepatic carcinogenesis [2].Conventional therapy modalities for HCC include surgical resection, liver transplantation, percutaneous ablation, and transarterial chemoembolization, and systemic chemotherapy CDK4/6 inhibition enhances T-cell immunotherapy on hepatocellular carcinoma cells by rejuvenating immunogenicity [3].In the past decade, immunotherapies, especially immune checkpoint inhibitors (ICIs) have emerged to bring the clinical management of advanced HCC to the next level.However, poor immune infiltration and dysregulated immunogenicity of cancer cells contribute to persistent immune suppression in solid tumors including HCC (known as 'cold tumor'), leading to a steep rise in treatment failures [4].Enhancing the immunogenicity of cancer cells and activating specific cytotoxic T cells are key factors for improving therapeutic efficacy against cancer.
Uncontrolled proliferation of cancer cells can be disrupted by cell cycle interference, which is indicated as a powerful treatment target for anti-cancer therapy.Accordingly, several selective inhibitors targeting cyclindependent kinase 4/6 (CDK4/6) have been applied to cancer treatment [5,6].Furthermore, cellular senescence, one of the steady cell-cycle arresting programs, mediates a dynamic state of cell fate transition.In malignant lesions, senescence and programmed cell death (PCD) can terminate the successive expansion of cancer cells intrinsically through cell cycle arrest [7].Nonetheless, unlike PCD, senescent cancer cells may restore immunogenicity not only through the senescence-associated secretory phenotype (SASP) but also by upregulating interferon γ-sensing and antigen-processing mechanisms to trigger adaptive T-cell surveillance [8,9].Last but not least, immune checkpoints (e.g., PD-L1) were found enriched in senescent liver cancer cells, mediating a suppressive tumor microenvironment and T-cell exhaustion, which can be partially reversed by immune checkpoint blockade [9][10][11][12].The complex role of senescence in anti-HCC treatment, especially in immunotherapies requires further investigation.
Here, we provide an explorative study to depict the comprehensive anti-cancer functions of CDK4/6 inhibition in HCC.According to the patient-derived liver cancer genomic data cohort, CDK4 and CDK6 were defined as key cell cycle regulators in HCC with high relevance to T-cell cytotoxicity and clinical outcomes.Interestingly, transcriptome analysis and in vitro investigations of CDK4/6 inhibition on HCC cells (HCs) revealed that CDK4/6-dependent cellular senescence broadly altered the levels of inflammatory factors and immune checkpoints in cancer cells.Taken together, our study elucidates a novel mechanism that CDK4/6 inhibition antagonizes senescence-related T-cell exhaustion via revitalizing immunogenicity in HCC.

Data source and analysis
Transcriptome datasets of liver cancers were obtained from the Cancer Genome Atlas Program (TCGA) and Gene Expression Omnibus (GEO) database (supplementary Table 1).Differentially expressed genes (DEGs) between groups were sorted out by limma package [13] with log (fold change, FC) > 1.5 and p < 0.05.Pathological immunohistochemistry images from liver tumor and non-tumor tissue samples were obtained from Human Protein Atlas (HPA) database.

Gene profiling in liver hepatic carcinoma (LIHC)
The Gene Expression Profiling Interactive Analysis (GEPIA) [14] was applied to investigate LIHC and normal liver tissue respectively based on gene profiling of TCGA [15] and the Genotype-Tissue Expression (GTEx) [16].We analyzed the expression levels of candidate genes in relation to overall survival (OS), disease-specific survival (DSS), progression-free survival (PFS), and disease-free survival (DFS).Survival time was analyzed and displayed using the Kaplan-Meier plotter.The mean follow-up time was above 10 years, and the Cox p-value was analyzed.The gene mutation landscape was analyzed to reveal related oncogene mutation by the ComplexHeatmap R package and plotted by the oncoprint [17].

Enrichment analysis and immune composition prediction
Function enrichment analysis was performed based on Gene ontology (GO) [18] and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases [19].The Tumor Immune Estimation Resource (TIMER) database was introduced to assess the main immune cell infiltration and related cancer outcome, based on the TCGA database [20].Cell composition of complex tissues from their gene expression profiles was characterized by the CIBER-SORT method [21].

Multiplex enzyme-linked immunosorbent assay (ELISA) detection
Supernatant samples were collected from cell culture at the end point of experiments.Concentrations of IFNγ, IL-1β, IL-6, and TNFα were detected using a Mouse Cytokine Panel 2 (4-Plex) purchased from Beijing Biolink Biotechnology Co.,Ltd (Beijing, China).The detection was performed following the manufacturer's instructions.Fluorescence reading was acquired using a microplate reader (Thermo Fisher, USA).

Senescence induction and detection
The HCC cells were treated with either Palbociclib or Doxorubicin in a concentration gradient for five days.The senescence-associated beta-galactosidase (SA-β-Gal) staining was performed according to the manufacturer's instructions.The SA-β-Gal + cells were imaged under a microscope (Olympus, Japan) and quantified using Fiji software (NIH Image, USA).The treatment-containing medium was replaced by a fresh complete medium once senescence was induced.

T-cell isolation and stimulation
C57BJ/6 mice were obtained from the animal core facility of Nanjing Medical University (Jiangsu, China).Animal experiments were approved by the Animal Welfare Committee of the affiliated Changzhou Second People's Hospital of Nanjing Medical University [Approval No. (2020) KYO 045 − 05].Mouse spleens were harvested and dissociated using a gentleMACS dissociator.The singlecell suspension was then filtered through a 40 μm mesh filter to remove possible cell clumps and centrifuged at 1,500 rpm for 5 min (4℃).The pellet was incubated with red cell lysis buffer for 10 min at room temperature.Then, the cell suspension was washed twice with pre-chilled 1× PBS and proceeded to pan-T cell isolation according to the manufacturer's instruction (Miltenyi, USA).The untouched T cells were stimulated with detachable CD3/CD28 Dynabeads™ in the complete T-cell medium supplemented with murine IL-2 (30 Units/mL) for 3 days according to the manufacturer's instructions of Dynabeads.

T-cell cytotoxicity assay and live-cell imaging
Pre-activated murine primary T cells were introduced and co-cultured with senescent HCC cells at a certain ratio (T-cell: HCC = 5:1).The T-cell cytotoxicity assays were determined by labeling damaged HCC cells using propidium iodide (PI) dye, which was monitored under the microscope (Olympus, Japan) for 72 h and assessed at the end point using a fluorescent microscope (Olympus, Japan).Protein levels of Ki67, γ-H2AX, and PD-L1 were detected using the immunocytochemistry protocol based on paraformaldehyde-fixed cells.Images were obtained under a fluorescent microscope (Olympus, Japan) and analyzed using Fiji software (NIH Image, USA).Antibodies and fluorescent dyes used in this study are listed in supplementary Table 2.

Flow cytometry
Mouse primary T cells were collected and centrifuged at 1,500 rpm for 5 min, followed by washing twice with 1× PBS.Then, cell samples were sequentially stained with a viability dye (Ghost Dye™ Violet) and primary conjugated antibodies according to the manufacturer's instructions.Data was obtained using a spectral flow cytometer (Canto II, BD, USA), and analyzed using FlowJo software 10.9 (BD, USA).Antibodies and fluorescent dyes used in this study are listed in supplementary Table 2.

Cellular calcium detection
Mouse primary T cells were seeded in 96-well plates.After the corresponding treatments, intracellular calcium was incubated following the instructions of the Fluo-4 NW Calcium Assay Kit (Thermo Fisher, USA).Simultaneously, cells were incubated with the Calmodulin-1/2/3 antibody after paraformaldehyde fixation.Fluorescence reading was acquired using a microplate reader (Thermo Fisher, USA).Cell numbers were estimated using a flow cytometer (Canto II, BD, USA).Fluorescence readings were normalized by the estimated live cell number of each group.

Gene expression quantification
Total RNA was extracted from cell homogenate using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA).Reverse transcription was conducted with a PrimeScript™ RT Reagent Kit (Takara Biotechnology Co., Ltd., Tokyo, Japan), and real-time quantification was subsequently performed using a SYBR® Premix Ex Taq Kit (Takara Biotechnology Co., Tokyo, Japan).The relative RNA expression levels were calculated using the -ΔΔCt method.Mouse primers included in this study are listed in supplementary Table 3.

Statistical analysis
GraphPad Prism 10.0 software (GraphPad Software, Inc., USA) and RStudio (Posit, USA) were used to generate plots.Significant differences were identified using an unpaired t-test.Pearson correlation analysis was performed to calculate the correlation coefficients."p < 0.05" commonly indicates a statistically significant difference.

CDK4/6 expression correlates with immune infiltration and outcomes of liver cancer
LIHC datasets were obtained from the TCGA database.Functional enrichment analyses (GO and KEGG) were conducted with upregulated DEGs (tumor vs. nontumor), showing that the cell cycle process was the most significantly enriched biological process (Fig. 1A and  B).Among all the CDKs, CDK1 and CDK4 were drastically overexpressed (tumor vs. non-tumor) (Fig. 1C).The OS and RFS of LIHC patients were correlated with low expression of CDK1, 2 and 4 (Fig. 1D).Hence, expression of CDK1, CDK2, and CDK4 demonstrated a significantly positive correlation with liver cancer stages (Fig. 1E and Supplementary Fig. 1A).In addition, we retrieved several published bulk RNA-sequencing datasets related to human HCC livers (GSE146719, GSE169289, GSE184733, GSE105130 and GSE166163).CDK4 expression was found significantly upregulated in tumor lesions from all datasets (Fig. 1F).Correlation of immune cell infiltration and CDKs (CDK1, CDK2, CDK4, and CDK6) was assessed from TCGA-LIHC and HCC datasets, which showed complex and contradictory results (Supplementary Fig. 1B and C).Protein-protein interactions between CDKs and immune checkpoints were also investigated and disclosed (Supplementary Fig. 1D).The interaction of CDK4/6 expression and immune cell infiltration [dendritic cells (DC), M0 / M1 /M2 macrophages and CD4 + /CD8 + T cells] in influencing the disease outcome of HCC patients was assessed, which showed that CD8 + T-cell infiltration with low CDK6 expression predicted the significant improvement of disease outcome (Fig. 1G and Supplementary Fig. 1E).In situ protein measurement based on immunohistochemistry (IHC) was obtained from HPA database.It showed that CDK4 and CDK6 protein levels increased in parenchymal areas (tumor vs. non-tumor) (Fig. 1H), while CDK1 and CDK2 remained undetectable (data not shown).Taken together, the cellcycle regulators CDKs (especially CDK4) play crucial roles in the disease prognosis of HCC.

Palbociclib and Doxorubicin enhance senescence and PD-L1 levels on HCs
To reveal the functions of CDK4/6 inhibition and DNA damage on HCs, Palbociclib (Palb) and Dox were introduced.Concentration-dependent cytotoxicity of Palb and Dox was titrated on human (HepG2) and mouse (Hepa1-6) HCC cell lines to select suitable concentrations (6 µM of Palb and 50 ng/mL of Dox) (supplementary Fig. 2A-D).Palb and Dox drastically suppressed proliferation of HepG2 and Hepa1-6 cells within 6 days, while Palb maintained cell numbers of Hepa1-6 from day 3 to day 6 (Fig. 2A and B).Simultaneously, cellular senescence induction by Palb and Dox was titrated on both cell lines, which suggested that Hepa1-6 responded better upon senescence induction (Supplementary Fig. 3A and  3B).Upon Palb or Dox treatment, cellular proliferation in Hepa1-6 was assessed through Ki67 staining (Fig. 2C) and the calculation of proliferation ratios (Fig. 2D), which showed that both Palb and Dox can drastically suppress HC proliferation.Furthermore, cellular senescence was detected by the accumulation of cytoplasmic SA-β-GAL (Fig. 2E, quantification is displayed in Fig. 2F) and nuclear γ-H2AX (Fig. 2G, quantification is displayed in Fig. 2H), the results of which showed that both Palb and Dox can significantly induce HC senescence.In addition, PD-L1 expression was upregulated on HCC cells after treatment with either Palb or Dox (Fig. 2I, quantification is displayed in Fig. 2J).Taken together, Palb and Dox were evidenced to induce cellular senescence and inflammatory cytokine production in HCs.

CDK4/6 inhibition-dependent HC senescence revitalizes T-cell cytotoxicity
To understand the efficacy of immunotherapies targeting senescent HCs, PD-1 or PD-L1 blockade combined with primary splenic T cells from strain-matched wildtype mice was introduced and co-cultured with either Palb-or Dox-induced senescent Hepa1-6 cells.T-cellmediated cytotoxicity against HCC cells was tracked and monitored for 96 h (Fig. 5A).After administration of HC-derived conditioned medium (CM), T cells were characterized by flow cytometry (Supplementary Fig. 6).The proportion of CD4 + T cells in total T-cell population increased significantly only after treatment with Dox-CM compared to vehicle CM, while CD8 + T-cell proportion decreased significantly after treatment with Dox-CM (vs.Palb-CM and vs. vehicle CM) (Fig. 5B).CD69 + cell proportions conventionally declined in T cells receiving CM treatment compared to the ex vivo expanded T cells as a positive control (Fig. 5C and D).CD69 + CD4 + T-cell proportion significantly increased in T cells treated with either Palb-CM or Dox-CM compared to vehicle CM (Fig. 5C), whereas CD69 + CD8 + T-cell proportion remained comparable in T cells receiving either Palb-CM  or Dox-CM (Fig. 5D).Simultaneously, the proportion of PD1 + CD4 + cells increased significantly in T cells treated with Palb-CM compared to both vehicle-CM-treated and the positive control T cells.(Fig. 5E).The proportions of PD1 + CD8 + cells increased significantly in all CM-treated T cells compared to the positive control T cells.Specifically, Palb-CM significantly increased the proportion of PD1 + CD8 + T cells in contrast to vehicle CM (Fig. 5F).Intracellular calcium activity assays indicated that both calcium intake and Calmodulin-1/2/3 protein levels were elevated by Palb-CM compared to vehicle CM, with the extent of this elevation even higher than that observed with Dox-CM, which evidenced active status of T cells (Fig. 5G).At the experiment endpoint, PD-1 and -L1 blockade didn't alter the T-cell-mediated cytotoxicity against untreated/proliferating HCs.Intriguingly, PD-1 blockade dramatically enhanced T-cell-mediated cytotoxicity against the Palb-induced senescent HCs, whereas PD-L1 blockade exhibited a limited T-cell derepressing effect in killing senescent HCs.In contrast, neither PD-1 nor PD-L1 blockade altered T-cell-mediated cytotoxicity against Dox-induced senescent HCC cells (Fig. 5H and  I).Taken together, CDK4/6 inhibition-dependent senescence on HCC cells activates T cells and prevents cytotoxic T cells from PD-1/PD-L1-mediated exhaustion.

Discussions
Despite various novel treatment modalities for HCC management, the prognosis of HCC patients is still poor.Less than 20% of HCC patients are eligible for surgical tumor removal, urging the development of efficient therapies, especially for advanced HCC.Practically, systemic chemotherapies (e.g., Doxorubicin) serve as indispensable therapies for HCC patients though, concern was arisen about uninspiring outcome improvement and serious toxic side effects [24].Trans-arterial chemoembolization and oral administration of targeted drugs (e.g., sorafenib) are currently most suggested in the clinical management of late-stage HCC patients [26].However, above two-thirds of HCC patients could not benefit from these treatments, notably followed by acquired drug resistance.The immune modulation process is regarded as a promising approach to enhance precisive attacks on cancer cells, which may be blunted by the suppressive microenvironment blocking the activation and function of cytotoxic immune cells in most solid tumors, including HCC.Therefore, singular immune-enhancing or immune checkpoint-blocking therapy exerts negligible efficacy on HCC [27,28].
T-cell surveillance is initially known as the framework to restrain chronic viral infection and pathogenic invasion [29].Tumor-specific or tumor-associated antigenpresenting processes occur in almost all cancer cells, to attract both helper and cytotoxic T cells.However, established cancer entities cannot be easily eliminated by autologous T cells in vivo.This is partially attributed to T-cell exhaustion, which can be induced by PD-L1 binding to PD-1 molecules on activated T cells [30].Later, with the discovery of various immune checkpoint proteins, PD-L1/PD-1 blockade gained a big victory in treating non-solid tumors, but it has posed a formidable challenge in the context of solid tumors [31].Accordingly, it offers a promising prospect to improve the efficacy of immune checkpoint blockade in combination with chemotherapies or targeted therapies.Nevertheless, the high utility of adjuvant immuno-chemotherapy/ targeted therapy remains challenging to acquire [32].In recent decades, emerging studies have evidenced that tumor cells exert the potential to rewire the suppressive tumor microenvironment.Inimitably, cellular senescence may play a crucial role in fueling tumor progression and influencing immune surveillance [33].The abundance and composition of SASPs vary from senescence-inducing approaches, exerting different regulatory effects on cancer cell immunogenicity and T-cell surveillance [34].
In this study, we characterized liver cancers from novel angles by conducting high-throughput transcriptome analysis on multiple HCC patient cohorts, identifying that cell-cycle regulators CDK4 and CDK6 significantly influence patients' outcomes.Interestingly, suppression of CDK4/6 may not only provoke inflammatory secretion of HCC cells but also improve CD8 + T-cell-associated survival time.We also mined out elevated interactions between T cells and CDK4/6 low HCC cells from singlecell RNA-seq datasets.Furthermore, we hypothesized that CDK4/6 suppression exaggerated the immunogenicity of HCC cells by the induction of cell-cycle arrestdependent senescence.Doxorubicin (a DNA damage inducer) was introduced on HCC cells to compare with Palbociclib (a selective CDK4/6 inhibitor).Both two drugs appeared to induce HC senescence and strengthen immune factor expression.Nonetheless, they upregulated PD-L1 expression on HCs.Intriguingly, Palbociclibinduced senescent HCC cells promoted functional T-cell activation as evidenced by PD-1 upregulation.To dissect the contradictory results, T-cell cytotoxicity assays were performed to assess the actual cancer cell killing effects of T cells under either Palbociclib or Doxorubicin pre-treatments, along with either PD-1 or PD-L1 inhibitor.Results indicated that both drugs could promote the T-cell-mediated killing efficacy to a certain extent though, only the presence of PD-1 blockade further improves the T-cell cytotoxicity.Promisingly, we evidenced that CDK4/6 inhibition can not only depress HC proliferation but also lead to cytotoxic T-cell activation via senescence induction in HCs.Superior to DNA damage induction, CDK4/6 inhibition drastically promotes tumor immunogenicity.Our study certainly reveals a novel mechanism of CDK4/6 inhibition in treating HCC.What's more, the immunogenicity-enhancing effect of CDK4/6 inhibition antagonizes senescence-related T-cell exhaustion, which implies promising therapeutic strategies to combine CDK4/6 inhibitors with immune checkpoint blockades for HCC.
Hence, the lack of in-vivo HCC models in this study unfortunately weakened the mechanistic exploration.Initially, we anticipated establishing a primary HCC mouse model with carbon tetrachloride (CCL 4 ) administration or a syngeneic mouse model transplanted with a highly proliferative HC line to investigate therapeutic efficacy and immune infiltration in vivo.Regrettably, CCL 4induced tumor lesions exhibited heterogeneous CDK4/6 expression, while transplanted HCC cells failed to form convincing tumor lesions with typical tumor microenvironment.Thus, neither Palbociclib nor Doxorubicin treatments could be effectively resumed on these models.Hopefully, this issue may be resolved using our tailored in-vitro Liver-on-a-chip model currently under development, which harbors multiple cell types with dynamic immune cell circulation to recapitulate the tumor microenvironment [35].Notably, cell-cycle regulation may act as a key metronome in reshaping tumor cell immunogenicity and modulating tumor immunity.For future perspectives, cellular senescence and SASPs appear to be promising therapeutic targets to protect against cancer.

Fig. 1
Fig. 1 CDKs characterized by bulk transcriptome analysis from HCC patient cohorts.(A) GO and (B) KEGG enrichment analyses based on significantly upregulated genes (LIHC, Tumor vs. Non-tumor) were displayed.(C) Gene expression of CDKs in non-tumor and tumor samples, as well as fold changes, was displayed.(D) Correlation between CDKs (CDK1, CDK2, CDK4, and CDK6) and survival time (OS and RFS) was displayed.(E) Correlation between CDKs (CDK1, CDK2, CDK4 and CDK6) and liver cancer stages was displayed.(F) Expression of CDK1, CDK2, CDK4, and CDK6 was demonstrated based on five liver cancer databases.(G) HCC outcome correlating with expression of CDK4/6 and immune cell infiltration was illustrated.(H) In situ expressions of CDK4 and CDK6 were depicted on liver tumor and non-tumor tissue samples."*" represents p < 0.05

Fig. 2
Fig. 2 Single-cell transcriptome analysis depicting CDK4/6 expression and immune interactions in human HCC samples.(A) Cell types (HCC, HSC, endothelial cell, B cell, T cell, and myeloid cell) and (B) HCC samples were clustered with UMAP.(C) Expression of CDK4 and CDK6 was illustrated.(D) The abundance of outgoing and incoming signals from variable cell populations was displayed.(E) Inflammatory signaling interactions were assessed among HCs, T cells, and myeloid cells.The thickness of the arrows represents interaction abundance